ONCOLYTIC ACTIVITY OF HUMAN ORTHOPNEUMOVIRUS IN CANCER CELL LINES.

Oncolytic virotherapy is an emerging biotherapeutic platform for selectively infecting cancer cells and triggering apoptosis in a number of malignant cells due to robust viral replication. Studies related to the oncolytic activity of human orthopneumovirus (hOPV) are conflicting. AIM: This study was designed to elucidate the possible role of hOPV in the modulation of cell growth and apoptosis in cancer cell lines including human epidermoid carcinoma (HEp-2), lung epithelial cell line (A549), and breast cancer cell line (MCF-7). MATERIALS AND METHODS: The oncolytic activity of hOPV on cancer cells was studied in vitro. The virus titers were determined by tissue culture infectious dose (TCID50/mL) in A549 cell. The cytotoxic effect of the virus on HEp-2, A549, and MCF-7 was determined using MTT and trypan blue dye exclusion test assays. hOPV in the infected cells was detected using real-time reverse transcription polymerase chain reaction (rRT-PCR) and indirect immunofluorescence (IIF) assays. The relative expression of apoptosis-related genes (CASP-3, -8, -9, Bax, Bcl-2, Bcl-XL, TP53, P21) during virus infection was estimated using rRT-PCR assay in comparison with the house-keeping gene (GAPDH). RESULTS: hOPV infection inhibited the growth of HEp-2, A549, and MCF-7 cells in a dose-and time-dependent manner. At a multiplicity of infection (MOI) of 5, hOPV reduced the viability of A549 cells to about 16%, HEp-2 to 22%, and MCF-7 to 28% (p = 0.001), while no significant inhibitory effect was observed when cells were infected at MOI of 1 and 2. hOPV mRNA and antigens were detected in infected HEp-2, A549, and MCF-7 cells by RT-PCR and IIF. Upon hOPV infection, expression of CASP-3, -8, -9, as well as Bax, TP53, and p21 mRNA increased while expression of Bcl-2, Bcl-xL anti-apoptotic genes decreased. In hOPV-infected A549 cells, the fold increase of CASP-8 and CASP-9, Bax, TP53, and P21 expression exceeded significantly compared to that in HEp-2 or MCF-7 cells. CONCLUSIONS: Our results provide evidence that hOPV could be a potential candidate for oncolytic virotherapy.

Naked DNA immunization with full-length attachment gene of human respiratory syncytial virus induces safe and protective immune response.

Development of potent vaccine for human respiratory syncytial virus (HRSV) that confers better protection than natural infection remains a global challenge. Vaccination with naked DNA is considered successful approach for the control of many viral diseases. In this study, the potential of DNA vaccination using full-length attachment gene of HRSV type A Saudi strain cloned in pcDNA3.1+ vector (pcDNA/GA) was evaluated in BALB/c mice. The expression efficiency of pcDNA/GA was first confirmed in HEp-2 cells on RNA and protein levels. Mice immunization with either pcDNA/GA or the positive control formalin-inactivated vaccine (FI-RSV) has generated significant serum antibody concentration in ELISA (7.31±0.418 and 9.76±0.006 µg/ml, respectively) with superior neutralizing activity. Similarly, both immunogens evoked robust HRSV-specific CD8+ T-cell response in ELISPOT assay compared to mice immunized with pcDNA3.1+ vector or saline (negative controls). Challenge of the immunized mice with the wild-type HRSV did not provoke clinical symptoms or mortality in any mice group. On the 7th day post-challenge, mice were euthanized and lungs were extirpated for evaluation of viral load, histopathological changes and cytokine profile. A significant diminish in the viral load and histology score were concluded in lungs of pcDNA/GA immunized mice compared to those immunized with FI-RSV and negative controls. The pulmonary cytokine profile of pcDNA/GA immunized mice displayed notable upregulation of Th1-associated cytokines while that of FI-RSV immunized mice exhibited high levels of Th2-associated cytokines. In conclusion, the DNA vaccine candidate pcDNA/GA has proven prominent efficacy and safety in mouse model, which encourages further evaluation in clinical trials.

Giant haemangioma of the liver with haemangiodudenal fistula: the first reported case in literature.

Giant liver haemangiomas are usually asymptomatic with normal liver function, which makes the course long and uneventful. The most commonly reported complications of giant haemangiomas are rupture with intraperitoneal haemorrhage that is either traumatic or non-traumatic, consumption coagulopathy, Budd-Chiari syndrome and congestive heart failure. We describe the first reported complications of a giant liver haemangioma as a fistula between the haemangioma and the gastrointestinal tract.

A Novel Mutation in the FECH Gene in a Czech Family with Erythropoietic Protoporphyria and a Population Study of IVS3-48C Variant Contributing to the Disease.

Erythropoietic protoporphyria (EPP), a chronic erythropoietic porphyria, is characterized by excess accumulation of protoporphyrin, particularly in erythroid cells. EPP inheritance is complex, almost always associated with two molecular defects. In most EPP patients, clinical expression requires coinheritance of a private ferrochelatase (FECH) mutation trans- to a hypomorphic FECH*IVS3-48C allele. This leads to a decrease of FECH activity below the critical threshold. This is characterized by cutaneous photosensitivity in early childhood such as itching, burning, swelling and redness in sun-exposed areas. Hepatic failure occurs in some patients (about 1-10 % of EPP patients), which may necessitate liver transplantation. We investigated a Czech family with two patients with manifested EPP in four generations. We found a novel mutation, c.84G >A, in the FECH gene in four individuals including proband and his mother (G84A transition in exon 2; p.W28*). Both clinically manifested probands inherited the hypomorphic IVS3-48C allele as well, while two clinically latent individuals with FECH mutation did not. To address the question whether the relatively low incidence of EPP in the Czech Republic might be due to lower frequency of the IVS3-48C allele, we screened for the frequency of the low expression allele in a control Czech (West Slaves) Caucasian population. Such study has not been performed in any Slavic population. Among 312 control individuals, there were no IVS3-48C/C (c.68-23C-T) homozygotes; 35 IVS3-48C/T heterozygous individuals were detected. The frequency of IVS3-48C allele was thus found to be 5.5 % in the Czech population, comparable to most West Caucasian populations.

Hepatoerythropoietic Porphyria Caused by a Novel Homoallelic Mutation in Uroporphyrinogen Decarboxylase Gene in Egyptian Patients.

Porphyrias are metabolic disorders resulting from mutations in haem biosynthetic pathway genes. Hepatoerythropoietic porphyria (HEP) is a rare type of porphyria caused by the deficiency of the fifth enzyme (uroporphyrinogen decarboxylase, UROD) in this pathway. The defect in the enzymatic activity is due to biallelic mutations in the UROD gene. Currently, 109 UROD mutations are known. The human disease has an early onset, manifesting in infancy or early childhood with red urine, skin photosensitivity in sun-exposed areas, and hypertrichosis. Similar defects and links to photosensitivity and hepatopathy exist in several animal models, including zebrafish and mice. In the present study, we report a new mutation in the UROD gene in Egyptian patients with HEP. We show that the homozygous c.T163A missense mutation leads to a substitution of a conserved phenylalanine (amino acid 55) for isoleucine in the enzyme active site, causing a dramatic decrease in the enzyme activity (19 % of activity of wild-type enzyme). Inspection of the UROD crystal structure shows that Phe-55 contacts the substrate and is located in the loop that connects helices 2 and 3. Phe-55 is strictly conserved in both prokaryotic and eukaryotic UROD. The F55I substitution likely interferes with the enzyme-substrate interaction.

Microcirculation in pregnancy.

The microcirculation, like all physiological systems undergoes modifications during the course of pregnancy. These changes aid the adaption to the new anatomical and physiological environment of pregnancy and ensure adequate oxygen supply to the fetus. Even though the microcirculation is believed to be involved in major pregnancy related pathologies, it remains poorly understood. The availability of safe and non-interventional technologies enabling scientists to study the intact microcirculation of the pregnant patient will hopefully expand our understanding. In this article we review the physiological changes occurring in the microcirculation during pregnancy and the role of the microcirculation in gestational related pathologies. We will also describe the available techniques for the measurement and evaluation of the microcirculation. Lastly we will highlight the possible fields in which these techniques could be utilized to help provide a clearer view of the microcirculation in the pregnant woman.

Managing international migration in developing countries.

PIP: This article summarizes the findings of 180 participants from 57 governments attending the UN's International Office of Migration's (IOM) Migration Seminar in April 1997 in Geneva. The teams of researchers represented the four developing world regions: sub-Saharan Africa; South Asia; the Arab Region; and Mexico, Central America, and the Caribbean. The seminar was part of IOM's research project on emigration dynamics in developing countries, which was begun in 1993. Researchers shared a common conceptual framework, which recognized the changing socioeconomic, sociopolitical, demographic, and ecological conditions in each country and subregion, the role of networks between people in sending and receiving countries, and the nature of entry restrictions. The research and workshop aimed to help policymakers in developed and developing countries. Conference delegates found the research framework acceptable despite the differences between regions and countries. Conference delegates agreed that the IOM research project was a unique forum for exchange of information and experience between sending and receiving countries. Many participants wanted IOM to provide technical assistance that would help countries manage migration. Delegates strongly desired international commitments to human rights for migrants. Delegates wanted better information exchanges, particularly interchanges of experience on policy measures among Governments, and the stronger inclusion of migrants in management. Returning migrants needed assistance with reintegration. The delegates made 12 recommendations about establishment of an effective system of information exchange, research on emigration dynamics and return migration, development of measures for managing flows that respect existing employment structures, new agreements, and reliable information for migrants on living conditions in host countries.^ieng